PROJECT SUMMARY We propose to refine and exploit powerful new markers and labeling systems to visualize multiple proteins or other biomolecules by electron microscopy (EM), correlated with light microscopy (LM). EM is one of the most powerful techniques to see cell structures below optical resolution, but has suffered from lack of generally applicable genetically encoded labels until our recent development of new EM-compatible markers such as miniSOG, a small flavoprotein that will do for EM what Green Fluorescent Protein did for LM, and APEX, an engineered ascorbate peroxidase. We have developed split-miniSOG, two fragments that do nothing separately, but when brought back together reversibly regenerate miniSOG and its fluorescence and photooxidative capability. Our newly developed split-miniSOG complementation system allows us to visualize intermolecular interactions at high resolution by EM. We have developed new strategies and techniques to improve the acquisition of element-specific analytical maps in transmission EM to achieve what we refer to as multicolor EM. This technology allows distinct EM- level labeling of multiple species with a different lanthanide element that is separately imaged by electron energy-loss spectrometry (EELS) and displayed in a distinct pseudocolor. Just as multicolor fluorescence has been vital to understanding many cellular functions at optical resolution, we anticipate multicolor EM will be valuable at finer resolution. Our overall goals are to expand and improve EM-compatible reporters, `molecular painting' chemistry, and new instrumentation to improve the resolution, sensitivity, and specificity with which multiple proteins or other biomolecules can be imaged by EM. We aim to obtain a genetically encoded far-red or near-infrared diaminobenzidine (DAB) photooxidizer analogous to miniSOG but excited at significantly longer wavelengths to facilitate multispecies labeling at high resolution. We will develop controlled living polymerization as an alternative to photooxidative amplification using modern methods such as ATRP, ROMP, or RAFT applied to fixed cells and tissue, to generate lanthanide-containing polymers of defined length and morphology at desired cellular targets. As test cases, these techniques will be applied to fundamental biological problems such as the spatial organization of the genome in the nucleus and aggregation of specified proteins involved in neurodegenerative diseases. We have chosen these biological processes because they are diverse, engage outstanding collaborators, and have great biomedical importance. Ultimately, the combination of photooxidizing, peroxidase-based, and nonphotochemical amplifying systems will give cell and molecular biologists a rich palette for EM, comparable to small-molecule fluorophores plus fluorescent proteins for optical microscopy.